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以干旱胁迫下斑茅(Saccharum arundinaceumRetz.)叶片组织的cDNA为Tester,正常生长条件下斑茅叶片组织cDNA为Driver,利用抑制性消减杂交技术(Suppression Subtractive Hybridization,SSH)构建斑茅干旱胁迫消减文库。文库克隆总量约为30 000个,克隆重组率为99.2%;随机从文库中挑取3500个克隆分析表明,插入片段长度主要集中在200~1 000 bp之间,500 bp以上的有463个克隆,500 bp以下的有3 037个克隆,平均长度约450 bp;通过随机对16个文库阳性克隆的测序及分析,3个克隆与ATP-NAD kinase、Aldo/keto reductase和锌指蛋白高度同源,3个克隆没有同源性,10个与逆境相关,是功能未知的EST。选择编号为EL0149和EL0145两个克隆进行Northern杂交验证,结果表明EL0149克隆为干旱胁迫上调表达的EST序列,而EL0145在正常供水和干旱条件下均无明显的的杂交信号。说明本研究克隆的SSH文库质量较高,可以进一步利用SSH文库结合芯片表达谱分析作物水分胁迫下的相关抗旱基因网络。
The cDNA of leaves of Saccharum arundinaceumRetz. Under drought stress was Tester. The cDNA of the leaves of Saccharomyces cernistris under normal growth conditions was Driver, and the cDNA library was constructed by Suppression Subtractive Hybridization (SSH) . The total number of clones was about 30 000 and the cloning and recombination rate was 99.2%. The analysis of 3500 clones randomly selected from the library showed that the length of the inserted fragments mainly concentrated in the range of 200-1 000 bp, and that of 463 The clones, 3 037 clones below 500 bp, with an average length of 450 bp. Three clones were highly homologous to ATP-NAD kinase, Aldo / keto reductase and zinc finger protein by sequencing and analysis of 16 library positive clones randomly The three clones had no homology, and ten were related to adversity. They were unknown ESTs. The results of Northern blotting showed that EL0149 was an up-regulated EST sequence under drought stress, while EL0145 had no obvious hybridization signal under normal water supply and drought conditions. This indicated that the SSH library cloned in this study is of high quality and can further utilize the SSH library in combination with the expression profile of the chip to analyze the related drought resistance gene networks under crop water stress.